High capacity assay platforms

ABSTRACT

A high capacity assay platform capable of binding target molecules includes a substrate and a polymer matrix attached to the substrate. The polymer matrix comprises a plurality of polymer molecules where at least some of the polymer molecules are covalently attached directly to the substrate and at least some of which molecules are crosslinked to other polymer molecules. Some of the polymer molecules have at least one binding ligand covalently attached thereto, and the density of the polymer matrix on the substrate is at least 2 μg/cm 2 .

REFERENCE TO RELATED APPLICATIONS

This application is a continuation of and claims the benefit of U.S.application Ser. No. 09/854,638, filed May 14, 2001, the disclosure ofwhich is hereby expressly incorporated herein by reference.

FIELD OF THE INVENTION

This invention relates to assay platforms for isolating, harvesting,detecting and/or quantitating target molecules, e.g., polypeptides,nucleotides or biomolecules, in or from a sample. More particularly, theinvention relates to multiwell assay plates and other assay platformscarrying a polymer matrix having a high density of binding ligandsdistributed therein.

BACKGROUND

A variety of approaches and techniques have been proposed and employedto provide assay platforms for high throughput, multi-sample screening.Multiwell plates have been treated, for example, to detect bindinginteractions. These assay plates have a relatively low density offunctional binding ligands. Consequently, the binding or capturecapacities of these assay plates are at sub-microgram levels andapplication potential is generally restricted to detection analysis.

Solid supports made of polystyrene, polypropylene and glass, such asmultiwell plates, glass slides, solid chromatography beads, sheets andtubes, are not suitable for the binding and isolation of multi-microgramamounts of high molecular weight target molecules per square centimeterincluding proteins, nucleic acids and polypeptides. To date no one hasbeen able to develop on a solid support a high density, high capacity,three dimensional structure that has the appropriate architecture forbinding large quantities of proteins and other molecular components.Attempts to covalently attach synthetic and natural polymers to supportshave not been successful in significantly increasing the bindingcapacity of the supports over that obtained with passive adsorption.

The challenge of isolating and identifying total protein expressed in anorganism in the rapidly growing field of proteomics requires advances intechnologies such as sample preparation, purification andcharacterization. Current methods for isolation of proteins and othermolecules require a considerable amount of effort, which generallyincludes employing time consuming chromatography or electrophoretictechniques. The current surface-derivatized multiwell plate systems lacksufficient surface area, porosity, and ligand density for the fastisolation of the microgram quantities needed for the characterization ofproteins, nucleic acids and other biomolecules.

Attempts by others to develop a method of rapidly and specificallyisolating multi-microgram amounts of proteins and other molecules persquare centimeter of surface from crude cellular extracts, allowingisolation and characterization of protein, have been unsuccessful.Previous failed attempts included covalently attaching a large amountand variety of natural and synthetic molecules to flat surfaces throughstandard organic or photochemical means.

SUMMARY OF THE INVENTION

The present invention provides an assay platform comprising a substrateand a polymer matrix attached to the substrate, wherein the polymermatrix is capable of binding target molecules, wherein the polymermatrix comprises a plurality of polymer molecules, wherein at least someof the polymer molecules are covalently attached directly to thesubstrate, wherein at least some of the polymer molecules arecrosslinked to other polymer molecules, wherein at least some of thepolymer molecules have at least one binding ligand covalently attachedthereto, and wherein the density of the polymer matrix on the substrateis at least 2 μg/cm².

The present invention also provides a method of preparing an assayplatform comprising a substrate and a polymer matrix attached to thesubstrate, wherein the polymer matrix is capable of binding targetmolecules comprising: contacting the substrate with a polymercomposition comprising a plurality of polymer molecules having repeatingunits, wherein at least some of the polymer molecules have at least onereactive group covalently attached thereto, wherein at least some of thepolymer molecules have at least one binding ligand covalently attachedthereto, wherein the polymer molecules have an average molecular weightof at least 100 kDa, and wherein at least 25% of the polymer moleculeshave at least one reactive group and at least one binding ligandcovalently attached thereto; and activating the reactive groups tocovalently bind at least some of the polymer molecules directly to thesubstrate and to induce cross-linking between polymer molecules to forma polymer matrix attached to the substrate.

The present invention also provides a polymer composition comprising aplurality of polymer molecules having repeating units, wherein at leastsome of the polymer molecules have at least one reactive groupcovalently attached thereto, wherein at least some of the polymermolecules have at least one binding ligand covalently attached thereto,wherein the polymer molecules have an average molecular weight of atleast 100 kDa, and wherein at least 25% of polymer molecules have atleast one reactive group and at least one binding ligand covalentlyattached thereto.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 compares the polypeptide binding capacity of an assay platformaccording to the invention to the polypeptide binding capacity of threecommercial plates.

DETAILED DESCRIPTION OF THE INVENTION

The high capacity assay platform of the invention overcomes the bindingcapacity limitations associated with currently available platforms. Aswill be described below, the present invention enables the rapid andspecific isolation of multi-microgram amounts of proteins and othertarget molecules, from even crude cellular extracts. The inventionenables one of skill in the art to characterize the isolated targetmolecule by standard biochemical methods, such as SDS PAGE, massspectrometry or Western blotting. A surprising and advantageous aspectof this invention is that a three dimensional polymer matrix comprisedof high density functionalized ligands and a controllable pore size canbe bound to a solid surface and used to isolate proteins andbiomolecules in a wide range of molecular weights. The porositycontrollable three dimensional matrix can be tailored to meet thespecific demands of the target.

The assay platforms of the invention may be prepared and used forisolating, detecting and/or quantitating a variety of target molecules,for example, peptides, polypeptides, large proteins, antibodies,glycoproteins, DNA, RNA and polysaccharides. As used herein, apolypeptide is any peptide having at least two amino acids. Apolypeptide may be a natural polypeptide or a synthetic polypeptide,e.g., recombinant or combinations thereof. The assay platforms of theinvention may also be prepared and used to bind small organic molecules,drugs, small proteins, peptides, chemically modified polypeptides,oligonucleotides or small polymers and compounds with smaller molecularweights. Selection of an appropriate pore size allows one of skill inthe art to advantageously develop a format that offers selectivity andhigh binding capacity.

The assay platforms of this invention comprise a substrate and a polymermatrix covalently attached to the substrate.

The substrate may be a material having a rigid or semi-rigid surface.The surface may be flat, curved and/or both. Additionally, the assayplatform of the invention may be provided in any desired form, size andshape. Examples of suitable substrates include plastic, glass,polystyrene beads, magnetic particles, other microparticles, polystyrenemultiwell plates, polypropylene multiwell plates, polycarbonatemultiwell plates, plasma-treated polystyrene surfaces, and Matrixassisted laser desorption ionization (MALDI) plates. MALDI massspectrometry is used for molecular weight determination. The method iscritical in the area of proteomics and is used to analyze proteolyticfragments or whole proteins obtained from crude or purified biologicalsamples. Samples are analyzed by MALDI mass spectrometry by placing thesamples at specific locations or spots on a MALDI plate. A MALDI plateis the solid support used to deliver the sample to the mass spectrometerand is also know as a MALDI target or MALDI probe capable of handlingirradiation by a laser during the analysis. Preferred assay platformsare multiwell plates, e.g., polystyrene or polypropylene multiwellplates containing 48, 96, 384 or 1536 individual wells.

As used herein, plastic is understood to be any of a group of syntheticor natural organic materials that may be shaped when soft and thenhardened, including many types of resins, resinoids, polymers, cellulosederivatives, casein materials, and proteins. Plastic materials, oftencalled resins, are made up of many repeating groups of atoms ormolecules linked in long chains (called polymers) that combine elementssuch as oxygen, hydrogen, nitrogen, carbon, silicon, fluorine, andsulfur. Both the lengths of the chains and the mechanisms that bond thelinks of the chains are related directly to the mechanical and physicalproperties of the materials. Characteristics of plastics materials canbe changed by mixing or combining different types of polymers and byadding nonplastics materials. Particulate fillers such as wood, flour,silica, sand, ceramic, carbon powder, tiny glass balls, and powderedmetal are added to increase modulus and electrical conductivity, toimprove resistance to heat or ultraviolet light and to reduce cost.Plasticizers are added to decrease modulus and increase flexibility.Other additives may be used to increase resistance to ultraviolet lightand heat or to prevent oxidation.

As used herein, glass is understood to be a brittle, noncrystalline,usually transparent or translucent material that is generally formed bythe fusion of dissolved silica and silicates with soda and lime. Glassis further understood to be any of a large class of materials withhighly variable mechanical and optical properties that solidify from themolten state without crystallization, that are typically based onsilicon dioxide, boric oxide, aluminium oxide, or phosphorus pentoxide,that are generally transparent or translucent, and that are regardedphysically as supercooled liquids rather than true solids.

The polymer matrix attached to the substrate comprises a plurality ofpolymer molecules wherein at least some of the polymer molecules have atleast one binding ligand covalently attached thereto. As used herein, abinding ligand shall be understood to mean a moiety that binds to atarget molecule by formation of either a covalent or noncovalent bondbetween the target molecule and the binding ligand. A covalent bond is astrong chemical bond between the binding ligand and the target moleculeby a sharing of electrons. A noncovalent bond is a weak chemical bondarising from nonspecific attractive forces of atoms oriented closetogether. The bonding between the binding ligand and target molecule maybe ionic or electrostatic, hydrogen bonding, or hydrophobic/hydrophilicinteractions or non-covalent.

The density of the polymer matrix on the substrate may be controlled by,inter alia, selection and amounts of the particular polymer and reactivegroups employed. The molecular weight of the polymer, the number andtype of reactive group and the number and molecular weight of thebinding ligands may be selected and adjusted, as detailed further belowand as illustrated in the Examples. The polymer matrix may be attachedto all of the substrate or to only a part of the substrate. For example,only the wells or a portion of the wells of multiwell plates may beprovided with the polymer matrix. Examples of other substrate include,beads completely covered by the polymer matrix.

Generally, the density of the polymer matrix on the substrate (totalmass of polymer, including covalently attached spacers and reactivegroups) is at least 2 μg/cm². In preferred embodiments, the polymermatrix has a density of 4 μg/cm² to 30 μg/cm², e.g., 6 μg/cm² to 15μg/cm².

Examples of suitable binding ligands which may be provided in thepolymer matrix include, but are not limited to: metal chelates, anionexchangers, cation exchangers, affinity ligands, hydrophobic bindingligands, chromatography ligands, chemically reactive ligands, covalentattachment sites, homobifunctional organic nucleophiles, polypeptides, apolynucleotide, an oligo dT, or a multivalent cation, wherein themultivalent cation is selected from the group consisting of copper,zinc, cobalt, gallium, iron and nickel. Illustrative binding ligandsinclude agonists and antagonists for cell membrane receptors, toxins andvenoms, viral epitopes, hormones, amino acids, peptides, polypeptides,enzymes, enzyme substrates, cofactors, drugs, lectins, dyes,nucleotides, phenylboronates, sugars, carbohydrates, biotin, avidin,streptavidin, oligonucleotides, nucleic acids, oligosaccharides,proteins, chelates, enzyme inhibitors, and antibodies. The proteinaffinity binding group may be streptavidin or avidin and the chelatormight be tetradentate nitriloacetic acid (NTA).

A metal chelator may be formed by the addition of a metal or a metaloxide to a chelating composition. Various metal chelators are currentlyavailable and may be used as a binding ligand of the invention. U.S.patent application Ser. No. 09/558,001, filed Apr. 24, 2000 and entitled“Metal Chelating Compositions,” discloses various suitable metalchelates and how they are produced, and is herein incorporated byreference. Additional metal chelators are known to those skilled in theart and include iminodiacetic acid, nitriloacetic acid or an analogthereof and diethylenetriamine pentacetic anhydride. In the mostpreferred embodiment of this invention the metal chelate is nickel,gallium, or iron.

An ion-exchanger may be a basic or acidic molecule covalently bound tothe polymer matrix that can interact or bind to molecules in solution,including macromolecules such as an enzyme, via charge interaction. Theion-exchange ligand may contain a nitrogen group, a carboxyl group, aphosphate group, or a sulfonic acid group. Examples of ion-exchangerbinding ligands include diethylaminoethyl (DEAE),diethyl[2-hydroxypropyl]aminoethyl (QAE), carboxymethyl (CM), andsulfopropyl (SP), and phosphoryl. See, Sigma Chemical Biochemicals andReagents 2000-2001 catalog, pages 1888-1899.

A hydrophobic binding ligand is a molecule covalently bound to thepolymer matrix possessing hydrophobic properties that can interact orbind to molecules in solution, including macromolecules such as anenzyme, via hydrophobic interaction. Examples of hydrophobic ligands arephenyl, hexyl, octly, and C18 groups. See, Sigma Chemical Biochemicalsand Reagents 2000-2001 catalog, pages 1936-1940.

A binding ligand can be a polypeptide, for example an antibody orproteins involved in protein-protein interaction. Alternatively, thebinding ligand can be any of the examples stated above which are capableof binding any target molecule including nickel, a polypeptide targetmolecule, a polynucleotide, or other biomolecules. A binding ligand canbe an amino acid, e.g., lysine, or a polypeptide having a molecularweight of 100 kDa or more, e.g., an IgG.

A binding ligand can be an oligonucleotide. The oligonucleotide bindingligand is capable of binding mRNA, cDNA or DNA and in turn may be usedto perform RT-PCR. An illustrative binding ligand is oligo dT.

The density of binding ligands in the polymer matrix may vary and may beselected or adjusted by changing the number and/or molecular weight ofthe ligands covalently attached to the polymer molecules of the matrix.Generally the density of binding ligands in the polymer matrix may be aleast 1 nanomole/cm². In some embodiments of the invention the densityof the binding ligands may be 1.2 nanomoles/cm² to 185 nanomoles/cm². Inanother embodiment of the invention the density of the binding ligandsmay be 1.5 nanomoles/cm² to 90 nanomoles/cm², e.g., 1.8 nanomoles/cm² to15 nanomoles/cm².

Through the selection and combination of various polymers and bindingligands, and by providing and controlling the cross-linking of polymermolecules in the matrix, the assay platforms of the invention enable thehigh capacity capture of target molecules in ranges not heretoforeachieved.

The polymer matrix of the assay platforms of the invention is capable ofbinding target molecules having a molecular weight of less than 3.5 kDain an amount of at least 1 nanomole/cm².

The polymer matrix may be constructed to be capable of binding targetmolecules having a molecular weight of 3.5 kDa to 500 kDa in an amountof 0.5 μg/cm² to 20 μg/cm², a molecular weight of 10 kDa to 500 kDa inan amount of 1 μg/cm² to 20 μg/cm², a molecular weight of 10 kDa to 350kDa in an amount of 2 μg/cm² to 20 μg/cm², a molecular weight of 10 kDato 350 kDa in an amount of 3 μg/cm² to 15 μg/cm². In some embodiments,the polymer matrix is capable of binding target molecules with amolecular weight of 10 kDa to 350 kDa in an amount of 4 μg/cm² to 10μg/cm². In certain embodiments the polymer matrix is capable of bindingpolypeptide target molecules having a molecular weight up to 350 kDa inan amount of at least 2 μg/cm² of polymer matrix.

The assay platforms of the invention may be prepared by contacting asubstrate with a polymer composition comprising a plurality of polymermolecules having repeating units, wherein at least some of the polymermolecules have at least one reactive group covalently attached thereto,wherein at least some of the polymer molecules have at least one bindingligand covalently attached thereto, wherein the polymer molecules havean average molecular weight of at least 100 kDa, and wherein at least25% of the polymer molecules have at least one reactive group and atleast one binding ligand covalently attached thereto. The reactivegroups are activated to covalently bind at least some of the polymermolecules directly to the substrate and to induce cross-linking betweenpolymer molecules to form a polymer matrix attached to the substrate.

The polymer molecules have repeating units that may be the same ordifferent and the polymer molecules may be linear or branched. Forexample, if the polymer molecules of the composition are proteins orpolypeptides, the repeating units would be amino acids. If the polymermolecules of the composition are carbohydrates, the repeating unitscould be glycosyl groups. Prefered polymers are non-polynucleotides.

The polymers may include several distinct polymer types, as prepared byterminal or side chain grafting. Some examples of distinct polymer typesinclude, but are not limited to, cellulose-based products such ashydroxyethyl cellulose, hydroxypropyl cellulose, carboxymethylcellulose, cellulose acetate and cellulose butyrate, acrylics such asthose polymerized from hydroxyethyl acrylate, hydroxyethyl methacrylate,glyceryl acrylate, glyceryl methacrylate, acrylic acid, methacrylicacid, acrylamide and methacrylamide, vinyls such as polyvinylpyrrolidone and polyvinyl alcohol, nylons such as polycaprolactam,polylauryl lactam, polyhexamethylene adipamide and polyhexamethylenedodecanediamide; polyurethanes, polylactic acids, linear polysaccharidessuch as amylose, dextran, chitosan, heparin and hyaluronic acid, andbranched polysaccharides such as amylopectin, hyaluronic acid andhemi-celluloses. Blends of two or more different polymer molecules canbe used. For example, in one embodiment the polymer molecules are amixture of dextran and heparin. In another embodiment dextran is mixedwith poly Lys-Gly (1 lysine per 20 glycine).

The polymers of the composition may be either natural or syntheticpolymers and modified natural or modified synthetic polymers. Thepolymers may also be dextran polymers. Natural polymers are branched orlinear polymers produced in a biological system. Examples of naturalpolymers include but are not limited to oligosaccharides,polysaccharides, peptides, proteins, glycogen, dextran, heparin,amylopectin, amylose, pectin, pectic polysaccharides, starch, DNA, RNA,and cellulose. A particular modified natural polymer that may be used isa dextran-lysine derivative produced by covalently inserting lysine intovariable linear positions along the dextran molecule using periodateoxidation and reductive amination or other methods known to those ofskill in the art.

Synthetic polymers are branched or linear polymers that are manmade.Examples of synthetic polymers include plastics, elastomers, andadhesives, oligomers, homopolymers and copolymers produced as a resultof addition, condensation or catalyst driven polymerization reactions,i.e., condensation polymerization.

Modified natural polymers are natural polymers that have been chemicallymodified. Chemical modifications can be done by, but are not limited to,oxidation, or the covalent attachment of photo-reactive groups, affinityligands, ion exchange ligands, hydrophobic ligands, other natural orsynthetic polymers, and spacer molecules.

Modified synthetic polymers are synthetic polymers that have beenchemically modified. Chemical modifications can be done by, but are notlimited to, oxidation, or the covalent attachment of photo-reactivegroups, affinity ligands, ion exchange ligands, hydrophobic ligands,orother natural or synthetic ligands.

The polymer molecules have an average molecular weight (total molecularweight of polymer, including covalently attached functional groups) ofat least 100 kDa, e.g., 300 kDa to 6,000 kDa. In some embodiments thepolymer molecules have an average molecular weight of 400 kDa to 3,000kDa. In another embodiment the polymer molecules have an averagemolecular weight of 500 kDa to 2,000 kDa. For purposes of this inventionthe average molecular weight is the weight average molar mass (Mw) valueof a polymer as measured by gel filtration chromatography usingmulti-angle light scattering and refractive index detection. The averageMw of the polymer distribution of all chain lengths present is basedupon the selection of the peak as measured by the refractive index,starting and ending peak selection criteria of a refractive index valuethat is three times the refractive index baseline. As shown by example apreferred polymer may have an average Mw of 1,117 kDa with a molecularweight range from 112 kDa to 19,220 kDa.

At least some of the polymer molecules of the composition contacted withthe substrate have at least one binding ligand covalently attachedthereto and at least some of the polymer molecules of the compositionhave at least one reactive group covalently attached thereto. As usedherein a reactive group is a chemical moiety that is capable ofcovalently bonding to the substrate. In addition, the reactive group mayalso be capable of covalently bonding to polymer molecules in thecomposition. This interaction of the reactive group between polymermolecule results in a cross-linking which forms the three-dimensionalmatrix. The reactive group reacts either thermochemically orphotochemically (polymers that contain a photo-reactive group arereferred to as being photolabeled).

Reactive groups include, but are not limited to, reactive groups used inthe preparation of chromatography media which include; epoxides,oxiranes, N-hydroxysuccinimide, aldehydes, hydrazines, maleimides,mercaptans, amino groups, alkylhalides, isothiocyanates, carbodiimides,diazo compounds, tresyl chloride, tosyl chloride, andtrichloro-S-triazine. For examples see, Methods in Enzymology Volume 34,Affinity Techniques, Enzyme Purification Part B, edited by William B.Jakoby and Meir Wilchek, Academic Press, New York, 1974, pp 1-809,hereby incorporated by reference.

Preferred reactive groups are α, β unsaturated ketone photo-reactivegroups. For purposes of this invention a photo-reactive group is amolecule or moiety that forms a highly reactive species upon exposure tolight. Examples of photo-reactive groups include aryl azides,diazarenes, beta-carbonyldiazo, and benzophenones. The reactive speciesare nitrenes, carbenes, and radicals. These reactive species aregenerally capable of covalent bond formation. Preferred photo-reactivegroups are photoactivatable, unsaturated ketones such as acetophenones,benzophenones and derivatives thereof. For examples see, LaboratoryTechniques in Biochemistry and Molecular Biology: PhotogeneratedReagents in Biochemistry and Molecular Biology, Hagan Bayley, Elsevier,New York, 1983, pp 1-187, hereby incorporated by reference.

The examples, which follow, depict a photo-reactive group that whencontacted with light becomes activated and is capable of covalentlyattaching to the surface of a solid substrate. For example thephoto-reactive groups may be activated by exposure to UV light fromabout 3 Joules/cm² to about 6 Joules/cm² depending on the intensity oflight and duration of exposure time. The exposure times may range fromas low as 0.5 sec/cm² to approximately 32 min/cm² depending on theintensity of the light source. In a preferred embodiment, thephoto-reactive groups are activated by exposure to light for 0.5 sec/cm²to 5.0 sec/cm² at about 1,000 mWatts/cm² to about 5,000 mWatts/cm², orfrom about 1,000 mWatts/cm² to about 3,000 mWatts/cm², e.g.,1,500mWatts/cm² to about 2,500 mWatts/cm².

There are many UV irradiation systems capable of delivering the totalenergy (dosage measured in Joules) required to bond the photo-activatedpolymer to a hydrocarbon rich substrate. Irradiation may be provided bya mercury lamp which has a distinct and known wavelength pattern ofirradiation. The intensity of irradiation requires Joules to fall in therange of 3-6 Joules/cm². Joule measurements encompass the time factor (1Joule=watt×second). In an embodiment of this invention the irradiationis provided by an electrodeless mercury lamp powered by microwaveradiation. One six inch, 500 watt/in. lamp has a rated power output of2,500 mWatts/cm² measured in the UVA range at about 2 inches distance oflamp to substrate. The lamp can be successfully run at 80% power orapproximately 2,000 mWatts/cm². Sample plates prepared using a standardlow intensity UV irradiation box having an intensity of irradiation(UVA/UVB, approximately 250 to 350 nm) measured at approximately 9.0mWatts/cm² and requiring greater than 10 Joules/cm² (10,000 mJoules)total energy to provide good bonding. This requires an incubation timeof the sample plates in the irradiation box of greater than 20 minutes.Plates processed using an electrodeless mercury lamp (2,000 mWatts/cm²)irradiation system requires only 1.75 sec/cm² for a total energy dosageof 3.5 Joules/cm². The higher intensity irradiation more efficientlyactivates the photo-active groups and consequently a lower overallenergy dosage is required.

Binding ligands and/or reactive groups may be covalently attached to thepolymer molecules via a spacer. For purposes of this invention a spaceris a molecule or combination of covalently bonded molecules that connectthe polymer molecule and either one or more of a binding ligand orreactive group. The spacer can be the same or different for any polymer,polymer composition or polymer matrix. Those of skill in the art willknow that many types of spacers are available and the selection and useis dependent upon the intended application of the polymer matrix, e.g.,a lysine molecule or a aminocaproic acid molecule.

The spacer can be covalently attached to the photo-reactive group by anumber of different chemistries including amide formation. For example,the use of the hydrocarbon spacer dramatically enhances polymer matrixstability performance. A photo-reactive group with a spacer may becoupled to a portion of a primary amine of the preferred polymer dextranby an amide bond at a controlled ratio relative to total monomer,glucose. For examples of spacers see the review by Jakoby and Wilchek,hereby incorporated by reference. Id at 1-809.

Examples of photo-reactive groups with a spacer include, but are notlimited to, benzobenzoic aminocaproic,N-Succinimidyl-N′-(4-azido-salicyl)-6-aminocaproate,N-Succinimidyl-(4-azido-2-nitrophenyl)-aminobutyrate, andN-Succinimidyl-(4-azido-2-nitrophenyl)-6-aminocaproate. Thesephoto-reactive groups with spacers may be reacted with a polymer toproduce a spacer that now includes the lysine as well as the originalspacer attached to the photo-reactive group. The spacer can also bemanufactured by incorporating multiple molecules such as lysine andaminocaproic acid prior to attaching the photo-reactive group containingor not containing an additional spacer. An example of a reactive groupcovalently attached to a polymer molecule is a spacer comprising amoiety or residue of lysine bound to one or more chemical entities ofthe reactive group, by the loss of a reactive hydrogen from the aminogroup.

The functioning of the polymer matrix is dependent on the spacing andnumber of binding ligands and reactive groups covalently attached to thepolymer molecules in the composition. This aspect of the invention isillustrated in the examples.

As illustrated, the density of primary amines contributed by the lysinespacers represents the density of desired binding ligand and reactivegroup. Modified polymers containing primary amines or other moietiessuch as spacers in a range of one moiety per every 1 to 100 polymerrepeating units may be made by procedures known in the art. Modificationof these moieties to selectively incorporate the desired amount ofreactive groups is also known. For example, the density of the primaryamines contributed by the lysine spacers is on average 1 for every 12repeating glucose units of the dextran polymer. This density is veryhigh relative to the desired incorporation of photo-reactive groups,e.g., less than one photo-reactive group per 200 repeating monomers. Theconcentration of primary amines in solution during polymer manufacturemight be 4.5 μmoles/mL, whereas the desired incorporation ofphoto-reactive groups would represent 0.09 μmoles/mL. Therefore, in thisinstance, there would be a 50-fold excess of primary amine to therequired photo-reactive group incorporation via a reactive ester. Atthis concentration of amine, employing the methodologies described inthe examples, the addition of photo-reactive group via a reactive esterat the desired level of incorporation results in greater than 90%efficiency of incorporation. By varying the amount of photo-reactivegroup containing a reactive ester any incorporation level less than 1reactive group per 200 monomers can be consistently achieved. The methodrequired to efficiently convert each of the remaining spacer moieties oramines to binding ligand attachment points is known in the art. Aseveral fold excess of an amine reactive, e.g., reactive ester,derivatization reagent is used for the attachment of the binding ligand,either directly in one step or through multiple steps. In some cases,the derivatization reagent will present an additional reactive groupwhich, depending on its reactivity, will dictate the stoichiometry forsubsequent binding ligand attachment. When lower ligand density isdesired the initial amine reactive derivatization reagent will belowered accordingly. In some instances free amines remaining afterselective modifications will generally be derivatized by acetylation.

When the polymer molecules have reactive groups covalently attached, thenumber of reactive groups is preferably less than 1 reactive group per200 repeating units. In another embodiment the polymer molecules haveless than 1 reactive group per 600 repeating units.

When the polymer molecules have binding ligands covalently attached theligands have from 1 binding ligand per 1 repeating unit to 1 bindingligand per 100 repeating units. In another embodiment the bindingligands covalently attached thereto have from 1 binding ligand per 1repeating unit to 1 binding ligand per 20 repeating units.

In a preferred embodiment of the invention the polymer moleculescontacted with the substrate have at least one binding ligand covalentlyattached thereto and at least some of the polymer molecules have noreactive group covalently attached thereto. The percentage of polymermolecules having both reactive groups and binding ligands covalentlyattached may be 25% to 80%. In another embodiment the percentage of bothreactive groups and binding ligands attached may be from 40% to 75%. Inyet another embodiment the percentage of both reactive groups andbinding ligands attached may be from 50% to 60%. In the preferredembodiment the percentage of polymer molecules having both reactivegroups and binding ligands covalently attached thereto may beapproximately 50%. The use of a mixture of polymer molecules, with andwithout reactive groups, enhances the highly functional formation of athree dimensional polymer matrix.

The first step in coating a surface of a substrate is contacting thepolymer composition with the substrate surface to be coated. The methodused to contact the polymer composition with the surface depends on thedimensions and shape of the surface to be coated. The surfaces can bemade, for example, from material selected from the group consisting ofpolystyrene, polypropylene, polyesters, polyethylene, silica, glass,latex, plastic, gold, iron oxide, polyacrylamide, nucleic acid, lipids,liposomes, synthetic polymers, proteins, polyaminoacids, albumins,antibodies, enzyme, streptavidin, peptides, hormones, andpolysaccharides. The surface can be derivatized prior to coating.Pre-derivatization can be done by any method known by one of skill inthe art, including silanization of silica and glass and plasma treatmentof polystyrene or polypropylene to incorporate amines, carboxyl groups,alcohols, aldehydes and other reactive groups or by chemicalmodification of the surface to change its chemical composition.

If necessary, the surface of the substrate may be chemically modified tofacilitate covalent bonding with the reactive groups carried on thepolymer molecules. Such modifications include treating the substratesurface with a hydrocarbon, or plasma-treating the surface. Anillustrative example of a chemical modification is the silanization ofglass. In a preferred embodiment a MALDI plate is dipped into a 1 mg/mLsolution of parafilm dissolved in chloroform and dried. [0053Whencoating a multiwell plate, tube or a surface or a portion thereof,larger than 0.1 mm square the polymer composition may be contacted withthe surface by pouring, mirco-pipeting, or transferring the polymercomposition onto the portions of the plate, i.e., wells to be coated. Inthe alternative, the portion of the plate, tube or a surface larger than2 mm square to be coated may also be coated by dipping the portion ofthe surface into a solution of the polymer composition so as to placethe surface in contact with the polymer composition. In the case ofsmaller surfaces, such as beads or chips, the surfaces can be dispersedinto a container possessing the polymer composition wherein the smallersurfaces are placed in direct contact with the polymer composition. Inaddition, once the beads or chips are placed into a container having thepolymer composition, the polymer composition containing the surfaces tobe coated can be stirred, agitated, or mixed to assure contact of thesurfaces to be coated with the polymer composition.

The amount of polymer that attaches to the solid surface may be adjustedor controlled by varying the polymer composition concentration andvolume added to the substrate. Once the polymer composition is placed incontact with the surface, the polymer composition may be dried on thesubstrate prior to activating the reactive groups, for example,evaporated to dryness by incubation in the dark at 20-50° Celsius withair flow. The polymer composition can also be evaporated usinglyophilization or by any other drying means, including air drying, toremove the solvent. A variety of drying methods may be used providedthat there is no premature activation of the reactive groups in responseto the drying step. The substrate is considered sufficiently dry when nomoisture is detectable visibly. During the drying the polymer moleculesof the polymer composition orient themselves so as to bind with thesubstrate surface or interact with each other to promote inter andintra-crosslinking with other polymers of the polymer composition.

The dried coated solid surface is then treated to induce the reactivegroups to covalently bond to the substrate. In the case of thephoto-reactive groups they may be activated by irradiation. Activationis the application of an external stimulus that causes reactive groupsto bond to the substrate. Specifically, a covalent bond is formedbetween the substrate and the reactive group, e.g., carbon-carbon bondformation.

In an embodiment activation may be done with a UVA/UVB light irradiatingat 9.0 mWatts/cm² for approximately 30 minutes to a total energy ofapproximately 15,000 mjoules/cm². In a preferred embodiment activationmay be done by exposure to UVA/UVB light irradiating at 2,000 mWatts/cm²to a total energy of from about 3 Joules/cm² to about 4 Joules/cm². Theamount of incubation time and the total energy used may vary accordingto the photo-reactive group bound to the polymer. In the most preferredembodiment, activation may be done by photoirradiation using a Fusion UVConveyor System using a mercury electrodeless lamp irradiating at 2,000mWatts/cm² with the conveyer belt set at 8 feet/minute with the lamppower at 400 watts/in. A radiometer, IL290 Light Bug, is run through theconveyer belt to verify the desired energy in the range of 3,000-4,000mjoules/cm². The multiwell plates are photoirradiated at about 800plates per hour, or about 1 plate per 4 to 5 seconds.

The concentration of the polymer composition of the present inventioncan be adjusted by changing the amount of total polymer per milliliterof solvent. In the case where a higher concentration of polymercomposition or polymer matrix per square cm would be advantageous, lesssolvent can be used to solvate the polymer molecules of the composition.In the case where a lower concentration of polymer composition orpolymer matrix per square cm would be advantageous, more solvent can beused to solvate the polymer molecules of the composition. In otherwords, adjusting the concentration of the polymer composition between0.02 and 1.0 mg/mL solvent and coating a solid surface such as amultiwell plate would produce a surface having a selectable range oftotal bound polymer matrix. The polymer composition can be completelysoluble or contain suspended insoluble polymer. The solvents that may beused to make the composition of the present invention include water,alcohols, ketones and mixtures of any or all of these. The solvents mustbe compatible with substrate being used. Since the polymers of thecomposition may crosslink between each other, it is possible that afluid-like solution of the composition may change into a gel. In thealternative, the solution may be produced in the form of a slurry.Examples of solvents that may be used in the composition include water,alcohols, ketones and mixtures of any or all of these.

Non-bound polymers may be removed by incubating in a suitable solutionto dissolve and remove unbound polymer. For example, multiwell platesmay be incubated with MOPS buffer overnight at 25° C., washed with MOPSbuffer and distilled water three times each, washed with hibitanesolution, air dried, packaged and stored below ambient temperature(2-820 C.). The remaining polymers form the polymer matrix.

Of the polymers that remain, at least some of the polymer molecules ofthe composition have at least one reactive group covalently attachedthereto. Some of the polymer molecules bind directly to the substratethrough the reactive group, whereas some reactive groups covalently bindbetween polymer molecules. The reactive groups are capable of covalentlybonding to more than one polymer molecule in the composition, theinteraction of the reactive group between polymer molecules results in across-linking which forms the polymer three-dimensional matrix.

If desired the binding ligands in the formed polymer matrix may bederivatized, e.g., by noncovalently or covalently attaching the bindingligands either by the addition of a different binding ligand or chemicalmodification of the existing binding ligand, thereby further enablingthe high capacity capture of a larger variety of target molecules. Thisbinding ligand modification feature of the invention is illustrated inExamples 1, 6, 7 and 9 below.

In preparing the assay platforms of the invention the substrate may becontacted with an amount of a polymer composition to provide a polymermatrix having a density of at least 2 μg/cm². In a preferred embodimentthe polymer composition is contacted with the substrate in an amountsufficient to provide a polymer matrix having a density of 4 μg/cm² to30 μg/cm², e.g., 6 μg/cm² to 15 μg/cm².

Additionally, an amount of the polymer composition may be contacted withthe substrate to provide a polymer matrix having a density of bindingligand of at least 1 nanomole/cm². In another embodiment the polymermatrix has a density of binding ligand of 1.2 nanomoles/cm² to 185nanomoles/cm². In the most preferred embodiment the polymer matrix has adensity of 1.5 nanomoles/cm² to 90 nanomoles/cm², e.g., 1.8nanomoles/cm² to 15 nanomoles/cm².

In one embodiment of the assay platform of the instant invention thesubstrate is a multiwell polystyrene plate, the polymer molecules aredextran polymers, the binding ligand is a nickel chelate and the polymermatrix has a binding ligand density of 1.5 nanomoles/cm² to 7.5nanomoles/cm². In other embodiments of the invention the binding ligandis a Gallium or Iron chelate or the binding ligand is glutathione.

In another embodiment of the invention the substrate is a multiwellpolypropylene plate, the polymer molecules are dextran polymers, thebinding ligand is an oligonucleotide. This binding ligand is preparedafter the original matrix is formed on the substrate. In many cases whena higher molecular weight ligand is added to the original ligand on thematrix the ligand density will decrease due to the larger size of thenew ligand.

In another embodiment of the invention the substrate is a multiwellpolystyrene plate, the polymer molecules are dextran polymers, thebinding ligand is streptavidin and the polymer matrix has a bindingligand density of 1.5 μg/cm² to 7.5 μg/cm². The binding ligand isprepared after the original matrix is formed on the substrate.

Finally, in another embodiment the invention has a substrate that is amultiwell polystyrene plate, the polymer molecules are dextran polymers,the binding ligand is selected from the group consisting of protein A,protein G, protein L or a mixture thereof, and the polymer matrix has abinding ligand density of 1.5 μg/cm² to 7.5 μg/cm². The binding ligandis prepared after the original matrix is formed on the substrate.

Produced examples described above in the assay platforms of theinvention can be used to isolate target molecules from solutionscontaining the target molecules. For example, a multiwell plate havingwells coated with the polymer matrix can be used to isolate targetmolecules from solution added to the individual wells of the plate. Oncethe solution is added to the plate, a period of time is allowed for thetarget molecule to react with the polymer matrix. As stated above theamount of time for reaction is a function of the target molecule,binding group, and the reason for using the plate. For example, if theplate is to be used as a quantitative measuring tool, the more time thatis allowed for the binding molecule to react with the target moleculethe greater the isolation of target molecule. If the plate is to be usedas a purification format then the more time allowed for the bindingmolecule to react with the target molecule the greater the amount oftarget molecule isolated for characterization. If the plate is beingused as a detection means, the amount of time that is allowed for thetarget molecule to react with the polymer matrix is less critical sinceit is the presence or absence of the target molecule that is important.

If the plate is being used as a quantitative measuring tool then theplate may be washed with water or a buffer after the target incubationperiod is completed. The amount of time before washing will varyaccording to the target molecule. In addition, the wash solution used,to remove unbound molecules from the plate, depends on the targetmolecule being isolated. For example, a hydrophobic solution would notbe used to wash the plate if a hydrophobic ligand was employed on thepolymer matrix, as it would remove the captured target molecule.

After washing, the target molecules that are covalently or noncovalentlyattached to the plate via the polymer matrix can either be disassociatedfrom the plate and removed for characterization or quantitation, left onthe surface to be detected using standard detection chemicals, or lefton the surface as the new binding ligand to further react withbiological or artificial samples to capture new target moleculesfollowed by detection or characterization. If the molecules are to bedisassociated from the plate the solvent used to disassociate the targetmolecules from the plate would depend on; the type of bonding betweenthe target molecules and the attached binding ligand of the polymermatrix, and the selected method of analysis or characterization. Forexample, if the bond is electrostatic in nature then washing the platewith a solution of a particular ionic strength or pH may disassociatethe target molecules from the plate. If the bond is hydrophobic innature then the molecules can be disassociated from the plate byreagents that break hydrogen bonding, e.g., urea. If the bond iscovalent in nature the target molecules can be disassociated from theplate by reagents and methods that break the covalent interaction.Dissociation may be accomplished by, but not limited to, chemical acidor base hydrolysis, proteolytic cleavage, and disulfide reduction.

If only detection of the target molecule is desired, the targetmolecules can be further reacted and detected while remaining attachedto the binding ligands of the polymer matrix. In other words, the targetmolecules do not need to be disassociated from binding ligand or removedfrom the substrate surface.

Various detection molecules can be reacted with the target molecules.Some detection molecules used include specific antibodies, eitherunmodified or modified to have a reporter molecule such as a fluorescentprobe or enzyme conjugates. The detection molecule used to react withthe target molecules bound to the plate depend on the nature of thetarget molecules. For example, if the target molecule is a protein, thena fluorescently labeled antibody can be used for detection. Othermolecules may use color changing molecules, e.g., antibody enzymeconjugates, to indicate the attachment to a bound target molecule. Oneskilled in the art would understand what type of molecule best could beused to detect the presence of the target molecule on the plate. Thetechniques for capture and analysis of target molecules are known to oneof skill in the art and examples are reviewed by Ed Harlow and DavidLane in, Antibodies, A Laboratory Manual, Cold Springs HarborLaboratory, 1998, pp 1-726, hereby incorporated by reference.

By way of example, if the presence of a particular protein in the urineof a patient indicates that the patient has a particular medicalcondition, a fluorescently labeled antibody that binds to the targetmolecule can be incubated on the plate after urine has been exposed tothe coated plate. If the target molecule was present in the urine, thenthat protein would bind to the binding ligand associated with thepolymer matrix on the plate. After washing the wells to remove theunbound material, the antibody is applied to the target molecule boundto the substrate. Placing the plate containing the target moleculefluorescent antibody complex, in UV light revels the target molecule fora means of quantitatively measuring the disease state. The same can beaccomplished using captured polynucleotides such as DNA and RNAfragments.

EXAMPLES Example 1

Preparation of a High Capacity Nickel Chelate 96 Well Plate

Preparation of Dextran-Lysine: Periodate oxidized dextran was preparedby adding 2.5 g (0.014 moles) of dextran (average molecular weight of3,368 kDa and range of 400 kDa to 54,000 kDa) into 31 mL of 0.05 Msodium acetate pH 5.0 buffer. The solution was stirred at roomtemperature until dissolved. The dextran was then cooled to 10° C. orlower in an ice bath. To the stirring dextran solution was added 3.6 mLof freshly prepared 0.45 M sodium periodate solution (1.7 mmoles). Thereaction mixture was protected from light and allowed to stir in an icebath for 2 hours. The periodate oxidized dextran solution was thenslowly added to 75 mL of a solution of 1.5 M lysine (112.5 mmoles)supplemented with 5.6 mL of 200 proof ethanol and 1 mL of pyridineborane under reduced light. The reaction mixture was allowed to stir for2 hours at room temperature while protected from light. Upon completionof the 2 hours, 5.83 mL of freshly prepared 2 M sodium borohydride wasadded to the reaction mixture and allowed to stir for 2 hours at roomtemperature. The reaction mixture was then transferred to dialysistubing for continuous dialysis against running water for a minimum of 18hours and then frozen and lyophilized to give a white fluffy solid (2.5g) with an average molecular weight of 696 kDa, with a molecular weightrange from 82 kDa to 11,000 kDa.

Preparation of Dextran-lysine-benzophenone: To 7.5 mL of 0.1 M sodiumphosphate pH 7.0 buffer was added 0.30 g (0.14 mmole of amine) ofdextran-lysine as prepared above and the mixture was stirred untilhomogeneous. 5.0 M hydrochloric acid was used to adjust the pH back to7.0 upon dissolution. 2.6 mL of N,N-dimethylformamide (DMF) was thenslowly added to the stirring solution, followed by 87 μL of a freshlyprepared solution of a 12 mg/mL benzobenzoyl aminocaproicn-hydroxysuccinimide (NHS) ester in anhydrous DMF. The reaction wasallowed to stir at room temperature while protected from light for 90minutes. The material was dialyzed against water and lyophilized to givea stable dextran-lysine-benzophenone intermediate (0.30 g).

Preparation ofDextran-lysine-benzophenone-bis(N,N-carboxymethyl)cysteine: To asolution of 0.30 g of dextran-lysine-benzophenone as prepared above,before lyophilization, was slowly added 750 μl of a freshly prepared 200mg/mL solution of maleimidobutyric acid NHS ester in DMF. The hazyreaction mixture was allowed to stir for 90 minutes at room temperature.To the stirring reaction mixture was added 63 μL of acetic anhydride.The reaction was stirred for 15 minutes at room temperature.Verification that all of the amines were blocked was determined using afluorescamine assay. A constant volume ultrafiltration wash wasperformed on the reaction mixture representing a 10-fold wash with 0.1 Msodium phosphate pH 7.0 buffer. The washed reaction mixture was bubbledwith argon for several minutes. To the washed reaction mix was added 351μL of a freshly prepared solution of 100 mg/mL of bis(N,N′-carboxymethyl)cysteine ligand in 0.1 M sodium phosphate pH 7.0buffer which was bubbled with argon. The reaction mixture was allowed tostir overnight at 2-8° C. while protected from light. The reactionmixture was dialyzed against running water overnight and thenlyophilized to give a white fluffy solid (0.30 g).

Preparation of Dextran-lysine-bis (N,N′-carboxymethyl)cysteine: To 0.3 gdextran-lysine as prepared above, before lyophilization was added 750 μLof a freshly prepared 200 mg/mL solution of maleimidobutyric acid NHSester in DMF was slowly added to the reaction mixture. The hazy reactionmixture was allowed to stir for 90 minutes at room temperature. To thestirring reaction mixture was added 63 μL of acetic anhydride. Thereaction was stirred for 15 minutes at room temperature. Verificationthat all of the amines were blocked was determined using a fluorescamineassay. A constant volume ultrafiltration wash was performed on thereaction mixture representing a 10-fold wash with 0.1 M sodium phosphatepH 7.0 buffer. The washed reaction mixture was bubbled with argon forseveral minutes. To the washed reaction mixture was added 351 μL of afreshly prepared solution of 100 mg/mL of bis(N,N′-Carboxymethyl)cysteine ligand in 0.1 M sodium phosphate pH 7.0buffer which was bubbled with argon. The reaction mixture was allowed tostir overnight at 2-8° C. The reaction mixture was dialyzed againstrunning water overnight and then lyophilized to give a white fluffysolid (0.3 g).

Preparation of High Capacity Nickel Chelate 96 well plate: To 4 columnsof a tissue culture treated (TCT) multiwell plate (96 well plate) wasadded 200 μL of a 0.15 mg/mL solution ofdextran-lysine-benzophenone-bis(N,N′-carboxymethyl)cysteine in water(100/0). To 4 additional columns on the same TCT 96 well plate was added200 μL of a 0.15 mg/mL in total dextran solution of 50%dextran-lysine-benzophenone-bis(N,N′-carboxymethyl)cysteine and 50%dextran-lysine-bis(N,N′-carboxymethyl)cysteine (50/50). The 96 wellplate was allowed to dry overnight in an oven at 40° C. with air blowingover it. The dried 96 well plate was then photoirradiated for 32 minutesat approximately 9.0 mWatts/cm². The plate was then soaked overnight in250 μL of water. The 96 well plate was then washed twice with 300 μL ofwater and loaded with 250 μL of a 3-(N-morpholino)butanesulfonic acid(MOPS) saline pH 7.0 containing 1 mM nickel sulfate hexahydrate andallowed to soak overnight at room temperature. After the target moleculenickel (59 daltons) is bound to the binding ligand the 96 well plateswere then washed once with 300 μL of 0.05 M acetic acid, once with 300μL of water, and then twice with 300 μL of MOPS pH 7.0 buffer.

Dextran Incorporation: The High Capacity Nickel Chelate 96 well plate,prepared as described above, was tested by determining the total dextranincorporation onto the 96 well plate. This data was achieved using ananthrone assay. To each section on the 96 well plate was added 200 μL ofan anthrone reagent. The 96 well plate was heated in an oven at 80-85°C. for 30 minutes to develop the color. The 96 well plate was read atA₆₂₀ against a dextran standard curve on the same 96 well plate in theblank wells to give the data as shown in table 1 below.

Protein Binding Capacity: The High Capacity Nickel Chelate 96 wellplate, prepared as described above, was also tested by determining thetotal amount of 50 kDa target protein, recombinant metal chelatingprotein containing FLAG fusion peptide (FLAG-Bacterial AlkalinePhosphatase (FLAG-BAP)) that could be captured on the 96 well plate. A0.15 mg/mL solution of a FLAG-BAP solution inTris(hydroxymethyl)aminomethane buffered saline (TBS) pH 8.0 wasincubated on the 96 well plate surface for 4 hours at room temperature.The FLAG-BAP was also substituted for either a 0.25 mg/mL solution of a15 kDa synthetic ribonuclease (RNAse)-6-His-biotin or a 0.225 mg/mLsolution of a 26 kDa histidine affinity tagged (HAT) fusiondihydrofolate reductase (DHFR) protein. After incubating for 4 hours,the 96 well plate was washed three times with 300 μL of phosphatebuffered saline with Tween 20 (PBST), followed by three washes with 300μL of water. A bicinchoninic acid (BCA) assay, using bovine serumalbumin (BSA) as a standard, was run on the 96 well plate to determinethe total amount of protein bound to the 96 well plate. The 96 wellplate was read at A₅₆₀ against a BSA standard curve on the same 96 wellplate in the blank wells to give the data as shown in table 1 below.

Purified FLAG-BAP (30 μg in 0.2 ml buffer per well) was added to allplates as described above. Bound FLAG-BAP was quantified directly onplate by BCA. Four different plates (A, B, C and D) were evaluated forprotein binding capacity. Plate A comprised the assay platform of thisinvention. Plates B, C and D were commercially available plates withimmobilized nickel used for capture, purification and detection ofhistidine containing proteins as follows: Plate B comprised Qiagen 35061Ni—NTA HisSorb Plate, Plate C comprised the Pierce 15142 Reacti-Bind™Metal Chelate Plate, clear strip plate and Plate D comprised the Pierce15143 Reacti-Bind™ Metal Chelate Plate, High Binding Capacity. Only theAssay Platform of this invention gave any detectable signal using theBCA assay which was 10 μg protein bound per well (6.7 μg protein/cm²).Plates B, C and D, the commercial plates, produced no detectable signalby which to evaluate protein binding capacity. The lower limit ofdetection was 0.3 μg protein/well which was equivalent to 0.2 μgprotein/cm². See, FIG. 1. TABLE 1 Dextran Loading and Protein BindingCapacity 50/50 100/0 Total Net Dextran, (μg) 7.30 8.17 μg dextran/cm²4.77 5.33 Expected Dextran, (μg/cm²) 2.67 5.33 % Increase Relative toExpected 178% A Dextran RNAse- Fusion 6-His- protein biotin HAT 50/50100/0 50/50 100/0 50/50 100/0 Total Net Protein, (μg) 6.42 5.00 4.51 ND3.57 ND μg protein/cm² 4.20 3.27 2.95 ND 2.33 ND Expected Total NetProtein, 1.64 3.27 ND ND ND ND (μg/cm²) % Increase Relative to 257% A NDND ND ND Expected Protein μg/cm² protein/μg/cm² dextran 0.88 0.61 0.62ND 0.49 ND‘A’ represents the normalized value of 100%.‘ND’ was information that was not determined at this time.

Specificity testing: To 4 wells in the 50/50 section of the 96 wellplate was added 200 μL of crude Escherichia coli (E. coli) extract(approximately 5 mg/mL in total protein) which contained the desiredrecombinant metal chelating protein, FLAG-BAP. To 4 wells in the 50/50section of the 96 well plate was added 200 μL of crude mammalian extractspiked to approximately 0.15 mg/mL with a recombinant metal chelatingprotein. To 4 wells in the same section of the 96 well plate was added200 μL 0.15 mg/mL recombinant metal chelating protein in TBS pH 8.0. Thewells were incubated overnight at 2-8° C. The 96 well plate was thenwashed 3 times with 300 μL PBST and 3 times with 300 μL of water. Toeach of the washed wells was added 200μL of a 0.1 M imidazole solutionin TBS pH 8.0. The 96 well plate was incubated on an orbital mixer for30 minutes at room temperature. The eluted proteins were assayed forpurity, both dilute and by a trichloroacetic acid (TCA) precipitation torepresent the protein bound in an entire well, by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE). The gel demonstrated thespecificity of binding from both E. coli and mammalian crude extractsbecause there was only a single band, equivalent to a purifiedrecombinant metal chelating control protein.

Example 2

Preparation of a Hydrophobic 96 Well Plate

Preparation of Dextran Diaminohexane: The periodate oxidized solution(1.25 g) prepared as in Example 1 was added to 30 mL of a solution of1.5 M diaminohexane, pH 8.5, supplemented with 5.6 mL 200 proof ethanoland 1 mL of pyridine borane. The reaction mixture was allowed to stirfor 2 hours at room temperature while protected from light. Uponcompletion of the 2 hours, 2.95 mL of freshly prepared 2 M sodiumborohydride was added to the reaction mixture and allowed to stir for 2hours at room temperature. The reaction mixture was then transferred todialysis tubing for continuous dialysis against running water for aminimum of 18 hours.

Preparation of Dextran-Diaminohexaneacetyl-Benzophenone: To 0.63 g ofdialyzed dextran-diaminohexane was added 5.5 mL of a 0.5 M SodiumPhosphate pH 7.0 buffer followed by 180 μL of a 12 mg/mL benzobenzoylaminocaproic NHS ester in anhydrous DMF with stirring. The reaction wasallowed to stir at room temperature while protected from light for 90minutes. To the stirring solution was added 400 μL (4.2 mmole) of aceticanhydride in 4 portions allowing 15 minutes of stirring in between eachaddition and a pH adjustment back to 7.0 with 5.0 M sodium hydroxide.Verification that all of the amines were blocked was determined using afluorescamine assay. The reaction mixture was then dialyzed againstrunning water overnight. The product was then frozen and lyophilized togive a white fluffy solid (0.6 g).

Preparation of Dextran-Diaminohexaneacetyl: To 0.63 g of the dialyzeddextran-diaminohexane solution was added 400 μL (4.2 mmole) of aceticanhydride in 4 portions allowing 15 minutes of stirring in between eachaddition along and a pH adjustment back to 7.0 with 5.0 M sodiumhydroxide. Verification that all of the amines were blocked wasdetermined using a fluorescamine assay. The reaction mixture was thendialyzed against running water overnight. The product was then frozenand lyophilized to give a white fluffy solid (0.6 g).

Preparation of a Hydrophobic 96 well plate: To 4 columns of a TCT 96well plate was added 200 μL of either a 0.15 mg/mL or a 0.5 mg/mLsolution of dextran-diaminohexaneacetyl-benzophenone in water (100/0).To 4 additional columns on the same TCT 96 well plate was added 200 μLof either a 0.15 mg/mL or 0.5 mg/mL in total dextran solution of 50%dextran- diaminohexaneacetyl-benzophenone and 50%dextran-diaminohexaneacetyl (50/50). To 4 additional columns on the sameTCT 96 well plate was added 200 μL of a 0.5 mg/mL in total dextransolution of 75% dextran-diaminohexaneacetyl-benzophenone and 25%dextran-diaminohexaneacetyl (75/25). The 96 well plate was allowed todry overnight in an oven at 40° C. with air blowing over it. The dried96 well plate was then photoirradiated for 32 minutes at approximately9.0 mWatts/cm². The 96 well plate was then soaked overnight in 300 μL ofwater. The 96 well plate was then washed twice with 300 μL of 0.3 Msodium chloride and twice with 300 μL of water.

Dextran Incorporation: The hydrophobic 96 well plate, prepared asdescribed above, was tested by determining the total dextranincorporation onto the 96 well plate. This data was achieved using ananthrone assay. To each section on the 96 well plate was added 200 μL ofan anthrone reagent. The 96 well plate was heated in an oven at 80-85°C. for 30 minutes to develop the color. The 96 well plate was read atA₆₂₀ against a dextran standard curve on the same 96 well plate in theblank wells to give the data as shown in table 2 below.

Protein Binding Capacity: The Hydrophobic 96 well plate, prepared asdescribed above, was also tested by determining the total amount oftarget protein that could be captured on the 96 well plate. A 1.0 mg/mLsolution of the target molecule, albumin 66 kDa in 1.0 M sodium sulfatewas incubated on the 96 well plate surface overnight at 2-8° C. The 96well plate was washed three times with 1.0 M sodium sulfate. (A negativecontrol 1.0 mg/mL solution of albumin in 0.005 M tris pH 8.0 wasincubated on the 96 well plate surface overnight at 2-8° C. The 96 wellplate was washed three times with 1.0 M Sodium Sulfate). A BCA assay wasrun on the 96 well plate to determine the total amount of protein boundto the 96 well plate. The 96 well plate was read at A₅₆₀ against a BSAstandard curve on the same 96 well plate in the blank wells to give thedata as shown in table 2 below. TABLE 2 Hydrophobic Plate 0.15 mg/mLdextran 0.5 mg/mL dextran load load 50/50 100/0 50/50 75/25 100/0 TotalNet Dextran, (μg) 3.91 6.26 3.779 9.109 19.360 μg dextran/(cm²) 2.564.09 2.47 5.95 12.65 Expected Dextran, (μg) 2.05 4.09 6.33 9.49 12.65 %Increase Relative to 125% A −61% −37% A Expected Dextran Total NetProtein (μg) ND ND 0.07 0.2 0.3 μg protein/(cm²) ND ND 0.046 0.13 0.20Expected Total Net Protein ND ND 0.10 0.15 0.20 (μg/cm²) % IncreaseRelative to ND ND −54% −13% A Expected Protein μg/cm² protein/μg/cm²dextran ND ND 0.019 0.022 0.022‘A’ represents the normalized value of 100%.‘ND’ was information that was not determined at this time.

Example 3

Preparation of a Dextran-Iminobispropylamine Anion Exchange 96 WellPlate

Preparation of Dextran-Iminobispropylamine: The periodate oxidizeddextran solution (1.25 g) prepared as above in example 1 was added to 30mL of a solution of 1.5 M, pH 8.5, iminobispropylamine supplemented with5.6 mL 200 proof ethanol and 1 mL of pyridine borane. The reactionmixture was allowed to stir for 2 hours at room temperature whileprotected from light. Upon completion of the 2 hours, 2.95 mL of freshlyprepared 2.0 M sodium borohydride was added to the reaction mixture andallowed to stir for 2 hours at room temperature. The reaction mixturewas then transferred to dialysis tubing for continuous dialysis againstrunning water for a minimum of 18 hours. The dialyzeddextran-iminobispropylamine was frozen and lyophilized to give a whitefluffy solid (1.25 g).

Preparation of Dextran-Iminobispropylamine-Benzophenone: To 44 mL of a14 mg/mL solution of dextran-iminobispropylamine in water was added 4.4mL of a 0.5 M sodium phosphate pH 7.0 buffer followed by the addition of180 μL of a 12 mg/mL bezobenzoyl aminocaproic NHS ester in anhydrous DMFwith stirring. The reaction was allowed to stir at room temperaturewhile protected from light for 90 minutes. The reaction mixture was thendialyzed against running water overnight. The product was then frozenand lyophilized to give a white fluffy solid (0.6 g).

Preparation of an Anion Exchange 96 well plate: To 4 columns of a TCT 96well plate was added 200 μL of either a 0.15 mg/mL or a 0.5 mg/mLsolution of dextran-iminobispropylamine-benzophenone in water (100/0).To 4 additional columns on the same TCT 96 well plate was added 200 μLof either a 0.15 mg/mL or 0.5 mg/mL in total dextran solution of 50%dextran-iminobispropylamine-benzophenone and 50%dextran-iminobispropylamine (50/50). To 4 additional columns on the sameTCT 96 well plate was added 200 μL of a 0.5 mg/mL in total dextransolution of 75% dextran-iminobispropylamine-benzophenone and 25%dextran-iminobispropylamine (75/25). The 96 well plate was allowed todry overnight in an oven at 40° C. with air blowing over it. The dried96 well plate was then photoirradiated for 32 minutes at approximately9.0 mWatts/cm². The 96 well plate was then soaked overnight in 300 μL ofwater. The 96 well plate was then washed twice with 300 μL of 0.3 Msodium chloride and twice with 300 μL of water.

Dextran Incorporation: The anion exchange 96 well plate, prepared asdescribed above, was tested by determining the total dextranincorporation onto the 96 well plate. This data was achieved using ananthrone assay. To each section on the 96 well plate was added 200 μL ofan anthrone reagent. The 96 well plate was heated in an oven at 80-85°C. for 30 minutes to develop the color. The 96 well plate was read atA₆₂₀ against a dextran standard curve on the same 96 well plate in theblank wells to give the data as shown in table 3 below.

Protein Binding Capacity: The Anion Exchange 96 well plate, prepared asdescribed above, was also tested by determining the total amount ofprotein that could be captured on the 96 well plate. A 1.0 mg/mLsolution of albumin in 0.005 M tris(hydroxymethyl)aminomethane (tris) pH8.0 incubated on the 96 well plate surface overnight at 2-8° C. The 96well plate was washed several times with 0.005 M tris. (A negativecontrol was a 1.0 mg/mL solution of albumin in 0.005 M tris pH 8.0containing 0.5 M sodium chloride incubated on the 96 well plate surfaceovernight at 2-8° C. The 96 well plate was washed several times with0.005 M tris pH 8.0.) A BCA assay was run on the 96 well plate todetermine the total amount of protein bound to the 96 well plate. The 96well plate was read at A₅₆₀ against a BSA standard curve on the same 96well plate in the blank wells to give the data as shown in table 3below. TABLE 3 Anion Exchange Plate 0.15 mg/mL dextran 0.5 mg/mL loaddextran load 50/50 100/0 50/50 75/25 100/0 Total Net Dextran, (μg) 1.631.25 1.62 1.36 1.39 μg dextran/(cm²) 1.07 0.82 1.06 0.89 0.91 ExpectedDextran, (μg/cm²) 0.41 0.82 0.45 0.68 0.91 % Increase Relative to 261% A233% 131% A Expected Dextran Total Net Protein (μg) 1.0  1.0  1.3  1.4 1.3  μg protein/(cm²) 0.65 0.65 0.85 0.92 0.85 Expected Total NetProtein (μg/cm²) 0.33 0.65 0.43 0.64 0.85 % Increase Relative to  50% A200% 144% A Expected Protein μg protein/μg dextran 0.61 0.80 0.80 1.030.94‘A’ represents the normalized value of 100%.‘ND’ was information that was not determined at this time.

Example 4

Preparation of a Dextran-Lysine Anion Exchange 96 Well Plate

Preparation of an Anion Exchange 96 well plate: To 4 columns of a TCT 96well plate was added 200 μL of either a 0.15 mg/mL or a 0.5 mg/mLsolution of dextran-lysine-benzophenone (as prepared in example 1) inwater (100/0). To 4 additional columns on the same TCT 96 well plate wasadded 200 μL of either a 0.15 mg/mL or 0.5 mg/mL in total dextransolution of 50% dextran- lysine-benzophenone and 50% dextran-lysine(50/50) (as prepared in example 1). To 4 additional columns on the sameTCT 96 well plate was added 200 μL of a 0.5 mg/mL in total dextransolution of 75% dextran-lysine-benzophenone and 25% dextran-lysine(75/25). The 96 well plate was allowed to dry overnight in an oven at40° C. with air blowing over it. The dried 96 well plate was thenphotoirradiated for 32 minutes at approximately 9.0 mWatts/cm². The 96well plate was then soaked overnight in 300 μL of water. The 96 wellplate was then washed twice with 300 μL of 0.3 M sodium chloride andtwice with 300 μL of water.

Dextran Incorporation: The anion exchange 96 well plate, prepared asdescribed above, was tested by determining the total dextranincorporation onto the 96 well plate. This data was achieved using ananthrone assay. To each section on the 96 well plate was added 200 μL ofan anthrone reagent. The 96 well plate was heated in an oven at 80-85°C. for 30 minutes to develop the color. The 96 well plate was read atA₆₂₀ against a dextran standard curve on the same 96 well plate in theblank wells to give the data as shown in table 4 below.

Protein Binding Capacity: The Anion Exchange 96 well plate, prepared asdescribed above, was also tested by determining the total amount ofprotein that could be captured on the 96 well plate. A 1.0 mg/mLsolution of the target molecule, albumin in 0.005 M tris pH 8.0incubated on the 96 well plate surface overnight at 2-8° C. The 96 wellplate was washed 3 times with 0.005 M tris pH 8.0. A negative controlwas a 1.0 mg/mL solution of albumin in 0.005 M tris pH 8.0 containing0.5 M sodium chloride incubated on the 96 well plate surface overnightat 2-8° C., the 96 well plate was washed several times with 0.005 M trispH 8.0. A BCA assay was run on the 96 well plate to determine the totalamount of protein bound to the 96 well plate. The 96 well plate was readat A₅₆₀ against a BSA standard curve on the same 96 well plate in theblank wells to give the data as shown in table 4 below. TABLE 4 IonExchanger 96 Well Plate 0.15 mg/mL dextran load 0.5 mg/mL dextran load50/50 100/0 50/50 75/25 100/0 Total Net Dextran, (μg) 3.36 5.08 3.479.11 12.93  μg dextran/cm² 2.20 3.32 2.27 5.95 8.45 Expected Dextran,(μg/cm²) 2.55 5.08 4.23 6.34 8.45 % Increase Relative to Expected 132% A−46%  −6% A Dextran Total Net Protein (μg) ND ND 0.9  1.0  2.8  μgprotein/cm² ND ND 0.59 0.65 1.83 Expected Total Net Protein ND ND 0.921.37 1.83 (μg/cm²) % Increase Relative to Expected ND ND −36% −53% AProtein μg/cm² protein/μg/cm² dextran ND ND 0.26 0.11 0.22‘A’ represents the normalized value of 100%.‘ND’ was information that was not determined at this time.

Example 5

Preparation of a Cation Exchange 96 Well Plate

Preparation of Dextran-lysine-benzophenone-succinylate: Dextran-lysine(0.63 g), as prepared in example 1, was dissolved in 44 mL of 0.05 Msodium phosphate pH 7.0 buffer. To the buffered dialyzeddextran-lysine-benzophenone solution was slowly added 180 μL of a 12mg/mL benzobenzoyl aminocaproic NHS ester in anhydrous DMF withstirring. The reaction was allowed to stir at room temperature whileprotected from light for 90 minutes. To the stirring reaction mixturewas added 4.2 mmole succinyl anhydride in 2 portions allowing 15 minutesof stirring in between each addition and pH adjustment back to 7.0 with5.0 M sodium hydroxide. A fluorescamine assay indicated that all of thefree amines on the dextran-lysine were not completely blocked with asuccinate group. An additional 1.6 mL of 100 mg/mL of succinyl anhydridewas added, stirred for 15 minutes and the pH was adjusted to 7.0. 100 μLof acetic anhydride was then added to the stirring reaction mixture andallowed to stir for 10 minutes. The pH was then adjusted back to 7.0 andthe fluorescamine assay indicated that the free amines were no longerpresent in the sample. The reaction mixture was then dialyzed againstrunning water overnight. The product was then filtered through 0.45micron filter, frozen, and lyophilized to give a white fluffy solid(0.60 g).

Preparation of Dextran-Lysine-succinylate: Dextran-lysine (0.63 g), asprepared in example 1, was dissolved in 44 mL of 0.1 M sodium phosphatepH 7.0 buffer. To this solution was added 4.2 mmole succinyl anhydridein 2 portions as above. A fluorescamine assay indicated that all of thefree amines on the dextran-lysine were not completely blocked with asuccinate group. An additional 1.6 mL of 100 mg/mL of succinyl anhydridewas added, stirred for 15 minutes and the pH was adjusted to 7.0. 100 μLof acetic anhydride was also added to the stirring reaction mixture andallowed to stir for 10 minutes. The pH was then adjusted back to 7.0 andthe fluorescamine assay indicated that the free amines were no longerpresent in the sample. The reaction mixture was then dialyzed againstrunning water overnight. The product was then filtered through 0.45micron filter, frozen and lyophilized to give a white fluffy solid(0.060 g).

Preparation of a Cation Exchange 96 Well Plate: To 4 columns of a TCT 96well plate was added 200 μL of either a 0.15 mg/mL or a 0.5 mg/mLsolution of dextran-lysine-benzophenone-succinylate in water (100/0). To4 additional columns on the same TCT 96 well plate was added 200 μL ofeither a 0.15 mg/mL or 0.5 mg/mL in total dextran solution of 50%dextran-lysine-benzophenone-succinylate and 50%dextran-lysine-succinylate (50/50). To 4 additional columns on the sameTCT 96 well plate was added 200 μL of a 0.5 mg/mL in total dextransolution of 75% dextran-lysine-benzophenone-succinylate and 25%dextran-lysine-succinylate (75/25). The 96 well plate was allowed to dryovernight in an oven at 40° C. with air blowing over it. The dried 96well plate was then photoirradiated for 32 minutes at approximately 9.0mWatts/cm². The 96 well plate was then soaked overnight in 300 μL ofwater. The 96 well plate was then washed twice with 300 μL of 0.3 Msodium chloride and twice with 300 μL of water.

Dextran Incorporation: The cation exchange 96 well plate, prepared asdescribed above, was tested by determining the total dextranincorporation onto the plate. This data was achieved using an anthroneassay. To each section on the 96 well plate was added 200 μL of ananthrone reagent. The 96 well plate was heated in an oven at 80-85° C.for 30 minutes to develop the color. The 96 well plate was read at A₆₂₀against a dextran standard curve on the same 96 well plate in the blankwells to give the data as shown in table 5 below.

Protein Binding Capacity: The cation exchange 96 well plate, prepared asdescribed above, was also tested by determining the total amount ofprotein that could be captured on the 96 well plate. A 1.0 mg/mLsolution of the 68 kDa target protein, avidin, in 0.005 M acetic acid pH5.0 incubated on the 96 well plate surface overnight at 2-8° C. The 96well plate was washed several times with 0.005 M acetic acid. A negativecontrol was a 1.0 mg/mL solution of avidin in 0.005 M acetic acid pH 5.0containing 0.5 M sodium chloride incubated on the 96 well plate surfaceovernight at 2-8° C. The 96 well plate was washed several times with0.005 M acetic acid pH 8.0. A BCA assay was run on the 96 well plate todetermine the total amount of protein bound to the 96 well plate. The 96well plate was read at A₅₆₀ against a BSA standard curve on the same 96well plate in the blank wells to give the data as shown in table 5below. TABLE 5 Cation Exchange 96 Well Plate 0.15 mg/mL dextran 0.5mg/mL load dextran load 50/50 100/0 50/50 75/25 100/0 Total Net Dextran,(μg) 2.75 5.95 3.59 4.52 11.27 μg dextran/(cm²) 1.80 3.89 2.35 2.95 7.37 Expected Dextran, (μg/cm²) 1.95 3.89 3.69 5.53  7.37 % IncreaseRelative to Expected −7% A −36% −47% A Dextran Total Net Protein (μg)2.8  6.1  6.3  8.3  17.5  μg protein/cm² 1.83 3.99 4.12 5.42 11.44Expected Total Net Protein (μg/cm²) 2.00 3.99 5.72 8.58 11.44 % IncreaseRelative to Expected −9% A −28% −37% A Protein μg protein/μg dextran1.02 1.03 1.75 1.84  1.56‘A’ represents the normalized value of 100%.‘ND’ was information that was not determined at this time.

Example 6

Preparation of a S-Acetylthioglycolic Acid Reactive 96 Well Plate

Preparation of Dextran-lysine-benzophenone-s-acetylthioglycolic acid :To 7.6 mL of 0.1 M sodium phosphate pH 7.0 buffer was added 0.30 g ofdextran-lysine as prepared in example 1 and the mixture was stirreduntil homogeneous. 5 M hydrochloric acid was used to adjust the pH backto 7.0 upon dissolution. 2.6 mL of N,N-dimethylformamide (DMF) was thenslowly added to the stirring solution followed by 87 μL of a freshlyprepared solution of a 12 mg/mL benzobenzoyl aminocaproicn-hydroxysuccinimide (NHS) ester in anhydrous DMF. The reaction wasallowed to stir at room temperature while protected from light for 90minutes. Upon completion of the 90 minutes, 775 μL of a freshly prepared200 mg/mL solution of s-acetylthioglycolic acid (SATA) NHS ester in DMFwas slowly added to the reaction mixture. The hazy reaction mixture wasallowed to stir for 90 minutes at room temperature. To the stirringreaction mixture was added 63 μL of acetic anhydride. The reaction wasstirred for 15 minutes at room temperature. Verification that all of theamines were blocked was determined using a fluorescamine assay. Thereaction mixture was dialyzed against running water overnight and thenlyophilized to give a white fluffy solid (0.30 g).

Preparation of Dextran-lysine-s-acetylthioglycolic acid: To 7.6 mL of0.1 M sodium phosphate pH 7.0 buffer was added 0.30 g of dextran-lysineas prepared in example 1 and the mixture was stirred until homogeneous.5.0 M hydrochloric acid was used to adjust the pH back to 7.0 upondissolution. 2.6 mL of N,N-dimethylformamide (DMF) was then slowly addedto the stirring solution followed by 775 μL of a freshly prepared 200mg/mL solution of SATA NHS ester in DMF was slowly added to the reactionmixture. The hazy reaction mixture was allowed to stir for 90 minutes atroom temperature. To the stirring reaction mixture was added 6 μL ofacetic anhydride. The reaction was stirred for 15 minutes at roomtemperature. Verification that all of the amines were blocked wasdetermined using a fluorescamine assay. The reaction mixture wasdialyzed against running water overnight and then lyophilized to give awhite fluffy solid (0.30 g).

Preparation of a S-Acetylthioglycolic Acid Reactive 96 well plate (A):To a TCT 96 well plate was added 200 μL of a 0.1 mg/mL solutiondextran-lysine-benzophenone-s-acetylthioglycolic acid in water (100/0).The 96 well plate was allowed to dry overnight in an oven at 40° C. withair blowing over it. The dried 96 well plate was then photoirradiatedfor 32 minutes at approximately 9.0 mWatts/cm². The 96 well plate wasthen soaked overnight in 300 μL of 10 mM MOPS containing 0.15 M sodiumchloride. The 96 well plate was then washed three times with 300 μL of10 mM MOPS containing 0.15 M sodium chloride and three times with 300 μLof water.

Preparation of a S-Acetylthioglycolic Acid Reactive 96 well plate (B):To 4 columns of a High Binding 96 well plate was added 200 μL of a 0.15mg/mL solution of dextran-lysine-benzophenone-s-acetylthioglycolic acidin water (100/0). To 4 additional columns on the same High Binding 96well plate was added 200 μL of a 0.15 mg/mL in total dextran solution of50% dextran-lysine-benzophenone-s-acetythioglycolic acid and 50%dextran-lysine-s-acetylthioglycolic acid (50/50). the 96 well plate wasallowed to dry overnight in an oven at 40° C. with air blowing over it.The dried 96 well plate was then photoirradiated for 32 minutes atapproximately 9.0 mWatts/cm². The 96 well plate was then soakedovernight in 300 μL of 0.025 M sodium acetate. The 96 well plate wasthen washed three times with 300 μL of 0.025 M sodium acetate and twicewith 300 μL of water.

Dextran Incorporation: The S-Acetylthioglycolic Acid reactive 96 wellplate (B), prepared as described above, was tested by determining thetotal dextran incorporation onto the 96 well plate. This data wasachieved using an anthrone assay. To each section on the 96 well platewas added 200 μL of an anthrone reagent. The 96 well plate was heated inan oven at 80-85° C. for 30 minutes to develop the color. The 96 wellplate was read at A₆₂₀ against a dextran standard curve on the same 96well plate in the blank wells to give the data as shown in table 6below. TABLE 6 S-Acetylthioglycolic Acid Reactive Plate 0.15 mg/mLdextran load 50/50 100/0 Total Net Dextran, (μg) 7.7 12.8 μgdextran/(cm²) 5.03 8.37 Expected Dextran, (μg) 4.19 8.37 % IncreaseRelative to Expected Dextran 120% A‘A’ represents the normalized value of 100%

Ligand Density: The SATA ligand density of the plate (B) was estimatedto be 1.56 and 2.60 nmoles/cm² for the 50/50 and 100/0 matricesrespectively.

Example 7

Preparation of the Streptavidin Coated 96 Well Plate

Preparation of the Streptavidin Coated 96 Well Plate (A): TheS-Acetylthioglycolic Acid reactive 96 well plate (A), as prepared inexample 6, was used to prepare a streptavidin coated 96 well plate. Toall of the wells on the coated 96 well plate was added 200 μL of a 0.1 Msolution of hydroxylamine in 0.1 M sodium phosphate pH 6.5. The 96 wellplate was incubated at room temperature for 5 minutes with rapid orbitalmixing. The 96 well plate was aspirated.

200 μL of a 0.1 mg/mL solution of the 60 kDa target molecule,streptavidin-maleimide in 0.1 M sodium phosphate pH 7.0 bubbled withargon was added to half of the S-Acetylthioglycolic Acid plate, 48wells, and 200 μL of a 0.05 mg/mL solution of streptavidin-maleimide wasadded to the remaining 48 wells. The 96 well plate was allowed toincubate in a cold room overnight to allow covalent attachment. Thesupernatant of several wells were combined and a Bradford assay wasperformed to determine that about 50% of the streptavidin-maleimide wascovalently attached to the plate. The binding was calculated to be 3.3and 6.67 μg streptavidin/cm² for the 0.05 and 0.1 mg/mL solutions,respectively. The plate was then washed 3 times with 300 μL of 0.1 Msodium phosphate pH 7.0 and 200 μL of 1.0 mM β-mercaptoethanol in 0.1sodium phosphate was added to react with free maleimide at roomtemperature for 15 minutes with orbital mixing. The plate was thenwashed three times with 300 μL of 0.1 M sodium phosphate pH 7.0 then 200μL of 1.0 mM bromoacetic acid in 0.1 M sodium phosphate pH 7.0 added andwas allowed to incubate for 1 hour at room temperature with orbitalmixing to block free sulfhydryls. The streptavidin plates were thenwashed 3 times with 300 μL of water and dried.

Preparation of the Streptavidin Coated 96 Well Plate (B): TheS-Acetylthioglycolic Acid reactive 96 well plate (B), as prepared inexample 6, was used to prepare a streptavidin coated 96 well plate. Toall of the wells on the coated 96 well plate was added 200 μL of a 0.05M solution of hydroxylamine in 0.15 M sodium chloride, 0.02 M sodiumphosphate, 0.001 M ethylenediaminetetraacetic acid (EDTA) pH 6.8. The 96well plate was incubated at room temperature for 45 minutes with rapidorbital mixing. The 96 well plate was aspirated.

200 μL of a 0.05 mg/mL solution of streptavidin-maleimide in 0.15 Msodium chloride, 0.02 M sodium phosphate, 0.001 M EDTA pH 6.8 was thenadded to each rows 2-4 and 8-10. The 96 well plate was allowed toincubate at room temperature with orbital mixing for 4 hours and then inthe cold room overnight. The supernatant of several wells were combinedand the A₂₈₀ was read as it compares to a buffer blank and to a solutionof 0.05 mg/mL streptavidin maleimide as loaded onto the 96 well plate.The results showed 4.18 and 4.38 μg streptavidin/cm² for 50/50 and100/0, respectively.

Protein Binding Capacity: To a streptavidin coated plate as preparedabove in (A) was added a 0.15 mg/mL solution of biotin-bovine serumalbumin (BSA) in PBS. The plate was allowed to incubate at roomtemperature for 1 hour with orbital mixing. A Bradford assay was run anddemonstrated 1.13 and 1.76 μg biotinylated albumin/cm² for the 3.3 and6.67 μg streptavidin/cm² plates, respectively.

Biotinylated Oligo Binding Capacity: Initially specificity of bindingwas determined by adding a 1.3 μM solution of5′-biotin-dT10-fluorescein-3′ in PBS to the streptavidin plates preparedabove (A) with varying concentrations free biotin and allowed toincubate for 15 minutes at room temperature. The plates were then washedand the fluorescence of any bound 5′-biotin-dT10-fluorescein-3′ wasdetermined on a plate reader. The results indicate that the free biotinwill inhibit the binding to the streptavidin plate demonstratingspecific affinity of the binding ligand. Secondly, variousconcentrations of 5′-biotin-dT10-fluorescein-3′ in PBS, ranging from0-10 μM were added to the plate in the absence of free biotin andallowed to incubate for 15 minutes at room temperature. The plates werethen washed and the fluorescence of any bound5′-biotin-dT10-fluorescein-3′ was read on a plate reader. The resultsindicated that the wells were saturated with5′-biotin-dT10-fluorescein-3′ at a concentration of approximately 2.5μM. The binding of the 5′-biotin-dT10-fluorescein-3′, the new bindingligand, was determined using anti-FITC antibody as the target molecule.

Example 8

Preparation of a Nickel Chelate Coated Matrix Assisted Laser DesorptionIonization (MALDI) Plate

Preparation of Nickel Chelate MALDI plate: A stainless steel MALDI platecontaining 20 individual sample spots, previously cleaned with methanol,was dipped into a 1 mg/mL solution of parafilm dissolved in chloroform.To the coated MALDI plate was added 3 μL of a 0.75 mg/mL in totaldextran solution of 50% dextran-lysine-benzophenone-bis (N,N′-carboxymethyl) cysteine (average molecular weight of 1,117 kDa andrange of 112 kDa to 19,220 kDa) and 50%dextran-lysine-bis(N,N′-carboxymethyl)cysteine (average molecular weightof 696 kDa and range of 82 kDa to 11,080 kDa) as prepared in example 1.The MALDI plate was allowed to dry for 30 minutes with air blowing overit. The dried MALDI plate was then photoirradiated for 32 minutes atapproximately 9.0 mWatts/cm² . The MALDI plate was then soaked with thetarget molecule by applying 3 μL of a 0.01 M nickel sulfate solution inwater for 30 minutes at 2-8° C. The target was stored in the cooler tohelp prevent evaporation. The MALDI plate was then soaked in water for20 minutes.

Protein capture a Nickel Chelate MALDI plate: The MALDI plate containingthe matrix with nickel chelate binding ligand was incubated with 3 μLper sample spot of a 0.5 mg/mL solution of the target molecule FLAG-BAPwith and without the presence of cude E. coli extract in TBS pH 8.0 for4 hours at 2-8° C. The MALDI plate was incubated in the cooler to helpprevent evaporation. The MALDI plate was then soaked in 0.01 M ammoniumbicarbonate for 20 minutes. The MALDI plate was dried in a MALDI platedrier. Sinapinic acid in 70% acetonitrile, 0.1% trifluoroacetic acid(TFA) was then added to each sample spot on MALDI plate and dried.Analysis was done using a Kratos Kompact Discovery SEQ MALDI-TOF massspectrometer. Results from the study indicate that the FLAG-BAP proteinwas detected and found to be pure whether captured from the purified orcrude samples. In addition, the FLAG-BAP protein was not detected on theMALDI plate that was soaked for 30 minutes in TBS pH 8.0 containing 100mM imidazole prior to the 0.01 M ammonium bicarbonate soak,demonstrating the specificity of the affinity capture.

Synthetic protein capture on a Nickel Chelate MALDI plate: The MALDIplate was incubated with 3 μL per sample spot of a 0.5 mg/mL solution ofa chemically synthesized 15 kDa target molecule, RNAse-6-His-biotin inTBS pH 8.0 for 4 hours at 2-8° C. The MALDI plate was stored in thecooler to help prevent evaporation. Upon completion of the 4 hourincubation time, the MALDI plate was soaked in 0.01 M ammoniumbicarbonate for 20 minutes. The MALDI plates were dried in a MALDI platedrier and then 1 μL sinapinic acid in 70% acetonitrile, 0.1% TFA wasthen added directly to each sample spot of the MALDI plate and dried.Analysis was done using a Kratos Kompact Discovery SEQ MALDI-TOF massspectrometer. Results from the study demonstrate that theRNAse-6-His-biotin was captured. In addition, the RNAse-6-His-biotin wasnot detected on the MALDI plate that was soaked for 30 minutes in TBS pH8.0 containing 100 mM imidazole prior to the 0.01 M ammonium bicarbonatesoak.

Peptide capture on a Nickel Chelate MALDI plate: The MALDI plate wasincubated with 3 μL per sample spot of a 0.1 mg/mL solution of a 3.2 kDasynthetic polypeptide target molecule, histidine containing peptide witha biotin tag in TBS pH 8.0 for four hours at 2-8° C. The MALDI plate wasstored in the cooler to help prevent evaporation. Upon completion of the4 hour incubation time, the MALDI plate was dipped in water to removeunbound material. To some of the sample spots on the MALDI plate wasadded 2 μL of a 0.1 mg/mL solution of streptavidin with and without 0.1mg/mL biotin in TBS pH 8.0. The MALDI plate was allowed to incubate at2-8° C. for 2 hours. The MALDI plate was then soaked in TBS pH 8.0containing 0.5 M sodium chloride for 20 minutes followed by 0.01 Mammonium bicarbonate for 20 minutes. The MALDI plate was dried in aplate drier and then 1 μL μ-cyano-4-hydroxycinnamic acid in 70%acetonitrile, 0.1% TFA was then added to the sample spots containingpeptide, sinapinic acid was used in place of α-cyano-4-hydroxycinnamicacid for spots containing peptide plus streptavidin. The MALDI plate wasdried. Analysis was done using a Kratos Kompact Discovery SEQ MALDI-TOFmass spectrophotometer. Results from the study indicate that the peptidewas detected in all cases, showing high affinity for the chelate bindingligand. In addition, streptavidin was detected in wells that wereincubated without the presence of free biotin showing that the newlyformed “chelate histidine containing peptide with a biotin tag” bindingligand has streptavidin affinity capture properties.

Example 9

Porosity Study of High Capacity Nickel Chelate Plate

Porosity study: A high capacity nickel chelate 96 well plate wasprepared, as give in example 1, using 0.2 mL of a mixture of one partdextran-lysine-benzophenone-bis (N,N′-carboxymethyl)cysteine and onepart dextran-lysine-bis(N,N′-carboxymethyl)cysteine (50/50) at a 0.2mg/mL and a 0.4 mg/mL in water. The average molecular weight of thepolymers was 737 kDa and range of 300 kDa to 10,000 kDa).

200 μL of a 0.15 mg/mL solution of FLAG-BAP protein was added to 9 wellsin each section of the plate and allowed to incubate in the cold roomovernight. The next morning, the plate was washed 3 times on anautomated plate washer with 300 μL of PBST. At that point, 200 μL of a25 μg/mL solution of a 150 kDa mouse monoclonal antibody, Anti-FLAG® M2(Sigma Product Code F3165), was added to 6 of the wells and allowed toincubate at room temperature for 5 hours to allow the antibody to bindto the FLAG-BAP protein, washed 3 times on an automated washer with 300μL of PBST. The Anti-FLAG® M2 step was repeated because the wells werenot saturated the first time due to high binding capacity. 200 μL of a100 μg/mL solution of Anti-FLAG® M2 was added and allowed to incubateovernight in the cold room. The plate was washed 3 times on an automatedplate washer with PBST. Finally, 200 μL of 100 μg/mL solution of a 150kDa anti-mouse IgG FITC conjugate was added to 3 of the wells thatcontained the protein protein interaction complex consisting of theFLAG-BAP protein and the Anti-FLAG® M2. This was allowed to incubate for2 hours at room temperature without shaking and 1 hour at roomtemperature with shaking to assure binding of the anti-mouse IgG FITCconjugate to the Anti-FLAG® M2. The plate was then washed 3 times on anautomated plate washer with 300 μL of PBST. The BCA reagent was allowedto develop for 1 hour at 37° C. and then the A₅₆₀ was determined on aplate reader. The overall results indicate that there was no restrictionin porosity up to 350 kDa.

Example 10

Preparation of the Anti-FLAG® M2 Coated 96 Well Plate

Preparation of—Anti-FLAG® M2 Maleimide Conjugate: To 1 mL of a 4.4 mg/mLsolution of Anti-FLAG® M2 in PBS was added 0.5 mL of 0.2 M sodiumphosphate buffer pH 6.7. To the stirring solution was added 10 μL of a0.03 μmoles/μL solution of maleimidocaproic acid NHS ester in anhydrousDMF. The solution was allowed to incubate for 1 hour at room temperatureand desalted on a Sephadex G50 column. The fractions were monitored byA₂₈₀ to give product in fractions 4-6, which gave 2.8 mL of product atapproximately 1.3 mg/mL.

Preparation of the Anti-FLAG® M2 Coated 96 Well Plate: To the SATAreactive 96 well plate (A), as prepared in example 6, was added 200 μLof a 0.1 M solution of hydroxylamine in 0.1 M sodium phosphate pH 6.5.The 96 well place was incubated at room temperature for 5 minutes withrapid orbital mixing. The 96 well plate was washed 3 times with 300 μLof 0.1 M sodium phosphate buffer pH 7.0 which was bubbled with argon.

200 μL of a 0.15 mg/mL solution of Anti-FLAG® M2 maleimide conjugate, asprepared above, 0.1 M sodium phosphate pH 7.0 which was bubbled withargon, was then added to each well in 2 columns on the plate. Thecolumns on the plate that were not incubated with the Anti-FLAG® M2maleimide conjugate were control wells for assaying purposes. The 96well plate was allowed to incubate at room temperature with orbitalmixing for 4 hours and then in the cold room overnight. The 96 wellplate was then washed 3 times with 200 μL of 0.1 M sodium phosphate pH7.0. 200 μL of a 1.0 mM β-mercaptoethanol solution in 0.1 M sodiumphosphate pH 7.0 was added to the wells that were previously incubatedwith Anti-FLAG® M2 maleimide conjugate and allowed to incubate for 15minutes at room temperature with orbital mixing. The plate was thenwashed 3 times with 0.1 M sodium phosphate buffer pH 7.0. To one columnthat was incubated with Anti-FLAG® M2 maleimide conjugate and one columnthat was not incubated with Anti-FLAG® M2 maleimide conjugate was loadedwith 200 μL of a 1.0 mM bromoacetic acid solution in 0.1 M sodiumphosphate pH 7.0 and allowed to incubate at room temperature for 1 hourwith orbital mixing. The wells that were not loaded with the bromoaceticacid solution were control wells for assaying purposes. The plates werethen washed 3 times with 300 μL of water.

Incorporation of Anti-FLAG® M2 on the 96 well plate: The Anti-FLAG® M2plate, prepared as described above, was tested by determining the totalprotein that was captured on the SATA reactive 96 well plate. A BCAassay was run to determine the amount of Anti-FLAG® M2 that was capturedon the plate with the remaining free sulfhydryl groups blocked and notblocked with bromoacetic acid, and against the wells that were notincubated with Anti-FLAG® M2 that were blocked and not blocked withbromacetic acid. The numbers generated in this assay were relative tousing BSA as a standard. The plates were read at A560. The results ofthe assay indicate that Anti-FLAG® M2 was incorporated to approximately3.90 and 4.04 μg/cm² over background for the unblocked and blockedrespectively.

Example 11

Preparation of a Poly dT Polymerase Chain Reaction (PCR) Plate

Preparation of Oligo-dT-30 C6-amine-Maleimide Conjugate: To a vialcontaining 0.5 μmoles of oligo dT-30 C6 amine (9.1 kDa) was added 0.5 mLof deionized water. The solution was then diluted 1:2 with 0.1 M sodiumphosphate pH 7.0. To the stirring, cloudy reaction mixture was added 21μL of a 10 mg/mL solution of maleimidobutyric NHS ester in anhydrousDMF. The reaction was allowed to stir at room temperature for 1.5 hours.A portion of the reaction mixture was removed and diluted to 0.01μmole/mL in PBS buffer pH 7.0 and supplemented to a final concentrationof 0.5 M sodium chloride. Several 5-fold serial dilutions were made togive concentrations of activated oligo dT 10,000, 2,000, 400, 80, and 16pmoles/mL.

Preparation of a Polypropylene Dextran-SATA 96 well PCR Plate: To a PCR96 well polypropylene plate was added 50 μL of a 0.15 mg/mL in totaldextran solution of dextran-lysine-benzophenone-s-acetylthioglycolicacid in water. The 96 well plate was allowed to dry overnight in an ovenat 40° C. with air blowing over it. The dried 96 well plate was thenphotoirradiated for 32 minutes at approximately 9.0 mWatts/cm². The 96well plate was then soaked overnight in 300 μL of 0.025 M sodiumacetate. The 96 well plate was then washed 3 times with 300 μL of 0.025M sodium acetate and twice with 300 μL of water.

Preparation of a Polypropylene Dextran-Oligo-dT 96 well PCR plate: Thedextran-SATA 96 well polypropylene PCR plate prepared as described abovewas loaded with 100 μL of 0.1 M hydroxylamine in 0.1 M sodium phosphate,pH 6.5, and allowed to incubate at room temperature for 15 minutes withorbital mixing. The plate was then washed 3 times with 300 μL of 0.1 Msodium phosphate pH 7.0 and loaded with 50 μL of the variousconcentration levels of the 9.1 kDa target molecule oligo dT-30C6-amine-maleimide conjugate, and allowed to incubate at 2-8° C.overnight with rapid orbital mixing. The 96 well plate was then washed 3times with 200 μL of 0.1 M sodium pH 7.0. 50 μl of a 1.0 mMβ-mercaptoethanol solution in 0.1 M sodium phosphate pH 7.0 was added tothe wells and allowed to incubate for 15 minutes at room temperaturewith orbital mixing. The plate was then washed 3 times with 0.1 M sodiumphosphate buffer pH 7.0. To each well of the plate was added 300 μL of a1 mM n-ethylmaleimide solution in 0.1 M sodium phosphate pH 7.0 andallowed to incubate at room temperature for 1 hour with orbital mixing.The plate was then washed 3 times with a 0.1 M sodium phosphate pH 7.0.The oligo dT plates were suitable for capturing mRNA and in turn usingthem to perform RT-PCR.

Example 12

Preparation of High Capacity Nickel Chelate 96 Well Plates Using HighThroughput Technology

Preparation of High Capacity Nickel Chelate 96 well plate: Using anOyster Bay dispenser, which has been first primed by dispensing at least10-fold the volume needed to fill a single 96 well plate, TCT 96 wellplates were filled with 200 μL of a 0.15 mg/mL solution of 50 %dextran-lysine-benzophenone-bis(N,N′-carboxymethyl)cysteine and 50 %dextran-lysine-bis (N,N′-carboxymethyl)cysteine, both as prepared inexample 1. The average molecular weight of the polymers was 1,104 kDawith a range of 300 kDa to 10,000 kDa). A check plate was made every 30minutes to be sure that the weight of the filled plate was within thespecified weight representing 200 μL of solution in each well. The 96well plates were placed on large drying trays and allowed to dry in adrying closet, in the dark overnight at 40-50° C. The dried 96 wellplates were then photoirradiated using a Fusion UV Converyor System withthe conveyer belt set at 8 feet/minute with the lamp power at 400watts/in. A radiometer, IL290 Light Bug, was run through the conveyerbelt to verify the desired energy in the range of 3,000-4,000mjoules/cm². The plates were photoirradiated at about 800 to 960 platesper hour. The 96 well plates were then soaked overnight in 250 μL ofwater, which was dispensed through the Oyster Bay. Again, using theOyster Bay, the 96 well plates were then washed twice with 250 μL ofwater and loaded with 250 μL of a 3-(N-morpholino)butanesulfonic acid(MOPS) saline pH 7.0 with 1 mM nickel sulfate hexahydrate buffer andallowed to soak overnight at room temperature. The 96 well plates werethen washed once with 300 μL of 0.05 M acetic acid, once with 300 μL ofwater, and then twice with 300 μL of MOPS-Hibitane pH 7.0 buffer. Theplates were then stacked and allowed to dry at room temperature prior touse.

Protein Binding Capacity: The High Capacity Nickel Chelate 96 wellplates, prepared as described above, were tested by determining thetotal amount of recombinant metal chelating protein, FLAG-BAP, thatcould be captured per well. A 0.15 mg/mL solution of a FLAG-BAP solutionin TBS pH 8.0 was incubated in the wells for 4 hours at roomtemperature. After incubating 4 hours, the wells were washed 3 timeswith 300 μL of PBST, followed by 3 times with 300 μL of water. A BCAprotein assay was run on a random sampling of wells from four differentplates to determine the total amount of protein bound per well. Resultsof the protein binding capacity were approximately 6 micrograms ofprotein per well or 4.2 micrograms protein per cm².

1. An assay platform comprising a substrate and a polymer matrixattached to the substrate, wherein the polymer matrix is capable ofbinding target molecules, wherein the polymer matrix comprises aplurality of polymer molecules, wherein at least some of the polymermolecules are covalently attached directly to the substrate, wherein atleast some of the polymer molecules are crosslinked to other polymermolecules, wherein at least some of the polymer molecules have at leastone binding ligand covalently attached thereto, and wherein the densityof the polymer matrix on the substrate is at least 2 μg/cm².
 2. Theassay platform according to claim 1 wherein the density of the polymermatrix on the substrate is 4 μg/cm² to 30 μg/cm².
 3. The assay platformaccording to claim 1 wherein the density of the polymer matrix on thesubstrate is 6 μg/cm² to 15 μg/cm².
 4. The assay platform according toclaim 1 wherein the polymer matrix has a binding ligand density of atleast 1 nanomole/cm².
 5. The assay platform according to claim 1 whereinthe polymer matrix has a binding ligand density of 1.2 nanomoles/cm² to185 nanomoles/cm².
 6. The assay platform according to claim 1 whereinthe polymer matrix has a binding ligand density of 1.5 nanomoles/cm² to90 nanomoles/cm².
 7. The assay platform according to claim 1 wherein thepolymer matrix has a binding ligand density of 1.8 nanomoles/cm² to 15nanomoles/cm².
 8. The assay platform according to claim 1 wherein thesubstrate is a multi-well plate.
 9. The assay platform according toclaim 8 wherein the multi-well plate is a 96, 384 or 1536 wellpolystyrene or polypropylene multiwell plate.
 10. The assay platformaccording to claim 1 wherein the substrate is a MALDI plate.
 11. Theassay platform according to claim 1 wherein the substrate is glass. 12.The assay platform according to claim 1 wherein the substrate isplastic.
 13. The assay platform according to claim 1 wherein the polymermolecules are natural polymers.
 14. The assay platform according toclaim 1 wherein the polymer molecules are dextran polymers.
 15. Theassay platform according to claim 1 wherein the polymer molecules aresynthetic polymers.
 16. The assay platform according to claim 1 whereinthe polymer matrix is capable of binding target molecules having amolecular weight of less than 3.5 kDa in an amount of at least 1nanomole/cm².
 17. The assay platform according to claim 1 wherein thepolymer matrix is capable of binding target molecules having a molecularweight of 3.5 kDa to 500 kDa in an amount of 0.5 μg/cm² to 20 μg/cm².18. The assay platform according to claim 1 wherein the polymer matrixis capable of binding target molecules having a molecular weight of 10kDa to 500 kDa in an amount of 1 μg/cm² to 20 μg/cm².
 19. The assayplatform according to claim 1 wherein the polymer matrix is capable ofbinding target molecules having a molecular weight of 10 kDa to 350 kDain an amount of 2 μg/cm² to 20 μg/cm².
 20. The assay platform accordingto claim 1 wherein the polymer matrix is capable of binding targetmolecules having a molecular weight of 10 kDa to 350 kDa in an amount of3 μg/cm² to 15 μg/cm².
 21. The assay platform according to claim 1wherein the polymer matrix is capable of binding target molecules havinga molecular weight of 10 kDa to 350 kDa in an amount of 4 μg/cm² to 10μg/cm².
 22. The assay platform according to claim 1 wherein the bindingligand is capable of binding a polypeptide target molecule.
 23. Theassay platform according to claim 1 wherein the polymer matrix iscapable of binding polypeptide target molecules having a molecularweight up to 350 kDa in an amount of at least 2 μg/cm².
 24. The assayplatform according to claim 1 wherein the binding ligand comprises ametal chelate.
 25. The assay platform according to claim 24 wherein themetal chelate is iminodiacetic acid, nitriloacetic acid or an analogthereof.
 26. The assay platform according to claim 1 wherein the bindingligand is capable of binding a polynucleotide target molecule.
 27. Theassay platform according to claim 1 wherein the binding ligand iscapable of binding mRNA target molecule.
 28. The assay platformaccording to claim 1 wherein the binding ligand is capable of binding aDNA target molecule.
 29. The assay platform according to claim 1 whereinthe binding ligand comprises a polynucleotide.
 30. The assay platformaccording to claim 1 wherein the binding ligand is covalently attachedto the polymer molecule through a spacer.
 31. The assay platformaccording to claim 30 wherein the spacer comprises a lysine molecule.32. The assay platform according to claim 30 wherein the spacer furthercomprises an aminocaproic acid molecule.
 33. The assay platformaccording to claim 1 wherein the substrate is a multiwell polystyreneplate, wherein the polymer molecules are dextran polymers, wherein thebinding ligand is a nickel chelate, and wherein the polymer matrix has abinding ligand density of 1.5 nanomoles/cm² to 7.5 nanomoles/cm². 34.The assay platform according to claim 1 wherein the substrate is amultiwell polystyrene plate, wherein the polymer molecules are dextranpolymers, wherein the binding ligand is a Gallium or Iron chelate, andwherein the polymer matrix has a binding ligand density of 1.5nanomoles/cm² to 7.5 nanomoles/cm²
 35. The assay platform according toclaim 1 wherein the substrate is a multiwell polystyrene plate, whereinthe polymer molecules are dextran polymers, wherein the binding ligandis glutathione, and wherein the polymer matrix has a binding liganddensity of 1.5 nanomoles/cm² to 7.5 nanomoles/cm².
 36. The assayplatform according to claim 1 wherein the substrate is a multiwellpolypropylene or polycarbonate plate, wherein the polymer molecules aredextran polymers and wherein the binding ligand is an oligonucleotide.37. The assay platform according to claim 1 wherein the substrate is amultiwell polystyrene plate or a multiwell polypropylene plate, whereinthe polymer molecules are dextran polymers, wherein the binding ligandis streptavidin, and wherein the polymer matrix has a binding liganddensity of 1.5 μg/cm² to 7.5 μg/cm².
 38. The assay platform according toclaim 1 wherein the substrate is a multiwell polystyrene plate, whereinthe polymer molecules are dextran polymers, wherein the binding ligandis selected from the group consisting of protein A, protein G, proteinL, or a mixture thereof and wherein the polymer matrix has a bindingligand density of 1.5 μg/cm² to 7.5 μg/cm².